The matrix metalloproteinases (MMPs) are associated with, and may be predictors of diseases such as osteoarthritis (OA), rheumatoid arthritis (RA), and cancer. In Phase I, we discovered novel and selective substrates for the MMP collagenase 3, an enzyme implicated in OA and cancer progression. Substrates were obtained by modifying sequence data gained from substrate mapping on protein expression libraries in phage lambda (phage display). We will extend this study to include other MMPs that are potential disease markers for not only OA and cancer, but also RA, so that we may obtain a profile of activities for a specific disease state. Phage display results are reported for stromelysin 1, gelatinase A and gelatinase B while a synthetic peptide library was used to determine the substrate specificity of collagenase 1. One goal is to use this knowledge to develop an "assay" kit that can measure these MMP activities directly in biological fluids. We will develop this kit by using two approaches: fluorescence substrates that are selective for the above MMPs, and neo-epitope antibodies to the protein fragments generated by collagenase 3 and/or gelatinase B cleavages. This "assay" kit can then be used in arthritis and cancer by assessing disease progression.